Nisa, I. C.; Chantachotikul, P.; Saito, T.; Bertocchi, C.; Deguchi, S.

Cellular senescence is characterized by stable cell-cycle arrest, cytoskeletal remodeling, and altered secretion of senescence-associated secretory phenotype (SASP) factors, including SERPINE1/plasminogen activator inhibitor-1 (PAI-1). Although extracellular matrix (ECM) stiffening has been linked to fibroblast mechanotransduction and SERPINE1-associated remodeling, the molecular pathway connecting substrate stiffness to SERPINE1 regulation in senescent fibroblasts remains incompletely understood. Here, we investigated how defined substrate stiffness affects fibroblast morphology, mechanical phenotype, and SERPINE1 expression, and examined whether the clathrin adaptor AP2A1 participates in this response in replicative senescent human fibroblasts. Using tunable polyacrylamide hydrogels, we found that increasing substrate stiffness enhanced fibroblast spreading, stress fiber thickening, focal adhesion maturation, cellular stiffness, and senescence-associated marker expression. Stiff substrates also increased SERPINE1 expression and its colocalization with actin fibers, with stronger responses observed in senescent than in young fibroblasts. Functional perturbation experiments further suggested that SERPINE1 contributes to stress fiber organization in senescent cells. In addition, AP2A1 colocalized with SERPINE1, and modulation of AP2A1 under knockdown and overexpression conditions altered SERPINE1 signal intensity. Conversely, perturbation of SERPINE1 also affected AP2A1, supporting a potential bidirectional relationship between these two components. Together, these findings identify SERPINE1 as a stiffness-responsive factor associated with senescence-linked cytoskeletal remodeling and support a functional relationship between AP2A1 and SERPINE1 in senescent fibroblasts. These results suggest that the AP2A1-SERPINE1 axis may contribute to the link between extracellular mechanical cues and senescence-associated fibroblast remodeling.